Genetic and pharmacological manipulation of system xc in pancreatic cancer

描述(申请人提供)：RAS是人类基因组中最有效的癌基因之一，在超过四分之一的人类癌症中发生突变[1]。特别是在胰腺癌中，KRAS以惊人的高突变率发生突变，超过95%的病例被激活[2]。虽然在胰腺癌转基因小鼠模型中同时消除RAS信号和突变的RAS蛋白表达可以完全切除肿瘤[3]，但事实证明，为胰腺癌患者开发特定的RAS抑制剂或其他有用的治疗方法几乎是不可能的。与这种缺乏进展相一致的是，胰腺导管腺癌(PDA)仍然是最致命的癌症之一，5年存活率仅为5%。显然，在胰腺癌领域开发新的治疗方法以寻找更有效和毒性更低的治疗方法是至关重要的。 目前已经做出努力，以确定其操纵可能被证明是致命的通路，特别是在含有RAS突变的细胞的背景下。这种“合成致死”组合可以在临床环境中提供巨大的效用，限制通常与放射和化疗方法相关的毒性，并以一种特定、有效的方式靶向RAS驱动的肿瘤。2003年，他进行了一次合成致命化学筛选。 斯托克韦尔发现了RAS突变细胞的特定杀手[4]。这种名为“erastin”的化合物通过抑制一种名为系统XC-[5]的半胱氨酸-谷氨酸逆向转运蛋白，导致一种铁依赖形式的氧化性细胞死亡，称为“铁下垂”。然而，这种方法在体内的实用性和XC-系统在RAS驱动的肿瘤中的确切作用仍不清楚。 鉴于突变的RAS在PDA的发生和维持中的作用，本研究的总体目标是验证系统XC功能在KRAS突变的胰腺癌的发生和维持中所必需的假说。具体地说，我们的目标是询问系统XC-的遗传和药物操作对胰腺癌细胞增殖和存活的影响。 利用分子遗传学、小鼠建模和临床前治疗方面的专业知识，我们将探讨System XC-在PDA生长和维持中的作用，并评估将这一途径作为胰腺癌患者治疗干预手段的临床可行性。具体地说，我们将使用shRNA和病毒技术在体外调节系统XC-。为了研究体内XC系统基因消融的效果，我们将XC系统功能缺陷的小鼠与我们的PDA临床前模型KPC小鼠进行杂交。最后，我们将使用一种从erastin中提取的化合物来评估系统XC-在体外和KPC模型中对胰腺癌细胞的药理抑制作用。 总之，本文提出的实验将有助于我们理解氧化还原状态维持在肿瘤生长和生存中的确切作用，并验证系统XC-抑制剂在胰腺癌有效治疗中的使用。

DESCRIPTION (provided by applicant): RAS is among the most potent oncogenes in the human genome and is mutated in over one in four of all human cancers [1]. In pancreatic cancer specifically, KRAS is mutated at an alarmingly high rate, activated in over 95% of all cases [2]. While extinguishing both RAS signaling and mutant RAS protein expression in a transgenic mouse model of pancreatic cancer completely ablates tumors [3], developing specific RAS-inhibitors or other useful therapies for pancreatic cancer patients has proven near impossible. In keeping with this lack of progress, pancreatic ductal adenocarcinoma (PDA) remains one of the most lethal cancers, with a 5-year survival rate of just 5%. It is clear that there is a critical ned to develop novel therapeutic approaches in the field of pancreatic cancer in order to identify more effective and less toxic treatments. Current efforts have been made to identify pathways whose manipulation could prove lethal specifically in the context of cells containing RAS mutations. Such "synthetic lethal" combinations could provide great utility in a clinical setting, limiting the toxicities commonly associated with both radiation and chemotherapeutic approaches, and targeting RAS-driven tumors in a specific, effective way. In 2003, a synthetic lethal chemical screen carried out by Dr. Stockwell identified a specific killer of Ras-mutant cells [4]. This compound, called "erastin," causes an iron-dependent form of oxidative cell death, termed "ferroptosis," through the inhibition of a cystine-glutamate antiporter named system xc- [5]. However, the utility of this approach in vivo and the precise role of system xc- in Ras-driven tumors remains unknown. Given the role of mutant RAS in the development and maintenance of PDA, the overall goal of the proposed research is to test the hypothesis that system xc- function is required for the development and maintenance of KRAS mutant pancreatic cancers. Specifically, we aim to interrogate the consequences of genetic and pharmacological manipulation of system xc- on the proliferation and survival of pancreatic cancer cells. Leveraging expertise in molecular genetics, mouse modeling, and preclinical therapeutics, we will interrogate the role of system xc- in the growth and maintenance of PDA and evaluate the clinical viability of targeting this pathway as a means of therapeutic intervention for pancreatic cancer patients. Specifically, we will use shRNA and viral technologies to modulate system xc- in vitro. To study the effects of genetic ablation of system xc- in vivo, we will cross mice deficient for system xc- function to our preclinical model of PDA, the KPC mouse. Finally, we will use a compound, derived from erastin, to evaluate the effect of pharmacological inhibition of system xc- on pancreatic tumor cells both in vitro and in the KPC model. In summary, the experiments proposed herein will aid our understanding of the precise role of redox state maintenance in cancer growth and survival as well as validate the use of system xc- inhibitors in the effective treatment of pancreatic cancer.